It is well accepted that most mutagens exert their biological effect by covalently binding to and altering the genomic DNA. However, there are a multitude of difficulties associated with specifying the chemical nature of the mutgenic DNA alteration, both because of the variety of DNA reaction products induced by many mutagens and because of the rarity of most mutational response. The goal of this proposal is to overcome these intrinsic problems by preparing DNA molecules containing chemically defined, biologically relevant damage at a specific location, to subject these templates to in vivo and in vitro replication, and to characterize the products both genetically and chemically. Using DNA from the well-defined M13 mp8/mp9 system, we will first prepare vectors capable of hybridizing to and ligating with site-specifically modified oligonucleotides. Next, we will extend our current studies on preparing oligonucleotides containing site-specific aminofluorenes, to include other adducts and other methods of preparation. Two approachs will be employed: chemical synthesis of oligonucleotides by the phosphotriester method using a specificlly modified nucleotide as one of the precursors, and nucleotide-specific reactions of various mutagens with preformed oligonucleotides. The types of DNA damage we will initially focus on include O6-ethylguanine, aminofluorene, and acetylaminofluorene benzo[a]pyrene. Site-specifically damaged M13DNA molecules will be transfected into appropriate strains of E. coli, representative mutants selected, and the relevant DNA sequences determined. Using this method we will be able to precisely correlate the chemical nature of a DNA lesion with a particular mutational event. The DNA molecules containing site-specific adducts will also serve as templates for DNA synthesis by several available DNA polymerases and in our well-characterized in vitro T7 DNA replication system. The availability of these templates will allow us to determine the precise effect these adducts have on both DNA synthesis by purified DNA polymerases alone and on the DNA replication process by a biologically relevant in vitro system.